Danielle M. J. Curfs

Macrophage secretory phospholipase A2 group X enhances anti-inflammatory responses, promotes lipid accumulation, and contributes to aberrant lung pathology

Secreted phospholipase A2 group X (sPLA2-X) is one of the most potent enzymes of the phospholipase A2 lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA2-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA2-X enzyme activity in vitro and in vivo, by generating sPLA2-X-overexpressing macrophages and transgenic macrophage-specific sPLA2-X mice. Our results show that sPLA2-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2). Moreover, we found that overexpression of active sPLA2-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA2-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA2-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA2-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA2-X in inflammatory lung disease.

Nuclear factor kappaB signaling in macrophage function and atherogenesis

Purpose of review Atherosclerosis is a chronic inflammatory disease of the medium and large-sized arteries. Nuclear factor kappaB transcription factors are major regulators of inflammatory responses, and aberrant nuclear factor kappaB regulation is linked to a large number of diseases. Focusing on macrophages, this review will discuss recent literature on the role of nuclear factor kappaB and the signaling pathways regulating its activity in atherosclerosis. Recent findings After the initial identification of activated nuclear factor kappaB in human atherosclerotic lesions, the involvement of this family of transcription factors in atherogenesis has gained growing attention. It is now clear that signaling pathways activating nuclear factor kappaB, and nuclear factor kappaB action, constitute major players at all stages of the atherosclerotic process. Long considered a pro-atherogenic factor, recent studies indicate that the actual role of nuclear factor kappaB might prove to be far more complex. Apart from activating many pro-inflammatory genes linked to atherogenesis, nuclear factor kappaB regulates cellular processes such as cell survival and proliferation. In addition, its important role in inflammatory resolution and anti-inflammatory gene transcription suggests that its activation at different cell types or different stages of the atherosclerotic process might have distinct and opposing results. Summary The numerous diseases in which aberrant nuclear factor kappaB action is found to play a crucial role makes it an intensively studied target for drug interventions. However, given its pleiotropic functions in inflammation and immunity, a more targeted modulation of its activity, at a cell type-specific or disease stage-specific level, could provide safer therapeutic solutions.

Benzo[a]pyrene Enhances Lipid Peroxidation Induced DNA Damage in Aorta of Apolipoprotein E Knockout Mice

The genotoxic compound benzo[a]pyrene (B[a]P) enhances atherosclerotic plaque progression, possibly by inducing oxidative stress and subsequent lipid peroxidation (LPO). Since LPO plays a key role in atherosclerosis, stable LPO derived DNA modifications such as 1,N6-ethenodeoxyadenosine (εdA) and 3,N4-ethenodeoxy-cytidine (εdC) may be useful biomarkers for in vivo oxidative stress. In this study, benzo[a]pyrene-diol-epoxide (BPDE)-DNA, εdA and εdC were determined by 32P-postlabelling in apolipoprotein E knockout (ApoE-KO) mice treated with 5 mg/kg B[a]P by gavage. After 4 days, BPDE-DNA adduct levels were higher in aorta (10.8±1.4 adducts/108 nucleotides) than in lung (3.3±0.7, P<0.05), which is a known target organ for B[a]P. Levels of εdA were higher in aorta of B[a]P-exposed animals than in unexposed controls (8.1±4.4 vs 3.4±2.1 adducts per 108 parent nucleotides, P<0.05). On the other hand, εdC levels were not affected by B[a]P exposure. Serum low density lipoprotein (LDL) levels were lower in B[a]P-exposed mice than in controls (9.3±3.7 and 13.3±4.0 mmol/l, respectively), whereas high density lipoprotein (HDL) levels were higher (1.4±1.6 and 0.4±0.3 mmol/l, respectively). Consequently, a three-fold difference in the LDL/HDL ratio was observed (P=0.001). εdA levels were positively related with plasma HDL concentrations (R=0.68, P=0.02), suggesting that the HDL mediated protection of the vessel wall against reactive lipid peroxides was reduced in B[a]P-exposed apoE-KO mice. Our observations show that direct as well as lipid peroxidation induced DNA damage is formed by B[a]P in aorta of apoE-KO mice, which may be involved in atherosclerotic plaque progression. This study further indicates that etheno-DNA adducts are useful biomarkers for in vivo oxidative stress in atherosclerosis.

Myeloid IκBα deficiency promotes atherogenesis by enhancing leukocyte recruitment to the plaques

Activation of the transcription factor NF-κB appears to be involved in different stages of atherogenesis. In this paper we investigate the role of NF-κB inhibitor IκBα in atherosclerosis. Myeloid-specific deletion of IκBα results in larger and more advanced lesions in LDL-R-deficient mice without affecting the compositional phenotype of the plaques or systemic inflammatory markers in the plasma. We show that IκBα-deleted macrophages display enhanced adhesion to an in vitro endothelial cell layer, coinciding with an increased expression of the chemokine CCL5. Also, in vivo we found that IκBαdel mice had more leukocytes adhering to the luminal side of the endothelial cell layers that cover the atherosclerotic plaques. Moreover, we introduce ER-MP58 in this paper as a new immunohistochemical tool for quantifying newly recruited myeloid cells in the atherosclerotic lesion. This staining confirms that in IκBαdel mice more leukocytes are attracted to the plaques. In conclusion, we show that IκBα deletion in myeloid cells promotes atherogenesis, probably through an induced leukocyte recruitment to plaques.

Environmental carcinogens and mutational pathways in atherosclerosis

Atherosclerosis is associated with DNA damage in both circulating and vessel-wall cells and DNA adducts derived from exposure to environmental mutagens are abundant in atherosclerotic vessels. Environmental chemical carcinogens identified as risk factor for atherosclerosis include polycyclic aromatic hydrocarbons (benzo(a)pyrene, dimethylbenz(a)anthracene, beta-naphthoflavone, pyrene, 3-methylcolanthrene), arsenic, cadmium, 1,3-butadiene, cigarette smoke. Accordingly, polymorphisms of genes encoding for phase I/II metabolic reaction and DNA repair are risk factor for cardiovascular diseases, although their role is negligible as compared to other risk factors. The pathogenic relevance of mutation-related molecular damage in atherosclerosis has been demonstrated in experimental animal models involving the exposure to chemical mutagens. The relevance of mutation-related events in worsening atherosclerosis prognosis has been demonstrated in human clinical studies mainly as referred to mitochondrial DNA damage. Atherosclerosis is characterized by the occurrence of high level of oxidative damage in blood vessel resulting from both endogenous and exogenous sources. Mitochondrial damage is a main endogenous source of oxidative stress whose accumulation causes activation of intrinsic apoptosis through BIRC2 inhibition and cell loss contributing to plaque development and instability. Environmental physical mutagens, including ionizing radiation, are a risk factor for atherosclerosis even at the low exposure dose occurring in case of occupational exposure or the high exposure doses occurring during radiotherapy. Conversely, the role of exciting UV radiation in atherosclerosis is still uncertain. This review summarizes the experimental and clinical evidence supporting the pathogenic role of mutation-related pathway in atherosclerosis examining the underlying molecular mechanisms.

Echogenic perfluorohexane-loaded macrophages adhere in vivo to activated vascular endothelium in mice, an explorative study

BackgroundMacrophages may concentrate ultrasound contrast agents and exhibit selective adhesion to activated endothelium. The present study investigates in mice the potential of perfluorohexane (PFH) loaded macrophages to act as ultrasound contrast agent with high reflectivity and specifically targeted at (atherosclerotic) vascular lesions.MethodsLung passage was evaluated with a mouse echo scanner after injection, at a slow pace or as a bolus, of varying doses of PFH-loaded and unloaded bone marrow macrophages (BMM) into the jugular vein. The interaction of PFH-loaded and unloaded BMM with TNF-α stimulated carotid artery endothelium after tail vein injection was assessed by means of intravital microscopy.ResultsHigh doses of jugular vein injected PFH-loaded BMM were visible with ultrasound in the pulmonary artery and detectable in the carotid artery. At intravital microscopy, tail vein injected BMM exhibited rolling and adhesion behavior at the TNF-α stimulated carotid endothelium, similar to that of native blood leukocytes. Rolling behavior was not different between PFH-loaded and unloaded BMM (p = 0.38).ConclusionIn vivo, perfluorohexane loaded macrophages pass the pulmonary circulation and appear on the arterial side. Moreover, they roll and adhere selectively to activated endothelium under physiological flow conditions. These findings indicate that perfluorohexane loaded BMM could be used to study processes in vivo where endothelial activation plays a role, such as atherosclerosis.